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. 2010 Jun 23;24(8):1543–1558. doi: 10.1210/me.2009-0381

Figure 1.

Figure 1

Uhmk1 is expressed in AtT-20 cells and affects POMC metabolism. A, Control AtT-20 cells or AtT-20 cells transiently expressing mycUhmk1 were subjected to Western blot analysis using the indicated Uhmk1 antibodies; arrows mark endogenous Uhmk1. B, pEAK Rapid cells transiently expressing mycUhmk1 were cotransfected with vectors (1 or 0.5 μg, as indicated) encoding Uhmk1 shRNA or two control shRNAs; Western blot analysis was performed using JH1998. Arrow, endogenous Uhmk1; *, nonspecific band. Quantification is shown below. C, Control AtT-20 cells or AtT-20 cells expressing Uhmk1 shRNA for 48 h were subjected to Western blot analysis using the indicated Uhmk1 or tubulin antisera; JH1998 AP, affinity-purified antibody; arrows, endogenous Uhmk1. D, Ratiometric analysis of AtT-20 and AtT-20 PAM-1 cells expressing mycUhmk1 (along with GFP in a dual promoter vector) vs. the GFP control or Uhmk1 shRNA/DsRed vs. the average of the two control shRNA/DsRed vectors. Fixed cells stained for cleaved ACTH or total POMC were visualized with Cy5-tagged secondary antibody. *, Ratios of samples with significant differences (P < 0.05 by t test and Kolmogorov-Smirnov). E, Representative AtT-20 PAM-1 cells expressing GFP and Uhmk1 from the dual promoter vector or dsRed/shUhmk1 24 h after transfection using antibody to ACTH; open arrows mark transfected cells; solid arrows mark nontransfected cells. Scale bar, 10 μm. Cont, Control.